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scfv antibody, encoding gene thereof and application thereof to preparation of preparation for treating or preventing infectious bursal disease

A gene and coding technology, applied in the field of scFv antibodies, can solve the problems of limitation and poor controllability of industrial production, and achieve the effect of strong specificity, avoiding horizontal transmission of diseases, and good therapeutic effect.

Active Publication Date: 2014-12-03
JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody drugs are currently effective therapeutic drugs. Hyperimmune serum and egg yolk antibodies can play a good role in the early stage of the disease, but they are limited due to poor controllability of industrial production and the existence of horizontal transmission of diseases.

Method used

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  • scfv antibody, encoding gene thereof and application thereof to preparation of preparation for treating or preventing infectious bursal disease
  • scfv antibody, encoding gene thereof and application thereof to preparation of preparation for treating or preventing infectious bursal disease
  • scfv antibody, encoding gene thereof and application thereof to preparation of preparation for treating or preventing infectious bursal disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Discovery of scFv antibody (single-chain antibody) and its coding gene

[0031] 1. Construction of antibody library

[0032] The spleens of chickens immunized with IBDV were taken, RNA was extracted and reverse transcribed into cDNA. According to the antibody sequence on GenBank, gene primers for cloning the variable region of the antibody were designed, and cDNA was used as a template to clone the variable region of the antibody by PCR. The VH and VL fragments were respectively inserted into the upstream and downstream of the Linker of the pTlinker vector to construct a scFv antibody library. The scFv antibody library was digested and connected to the bacterial display vector pBSD to construct a bacterial surface display library of anti-IBDV antibodies. VH is about 380bp, VL is about 320bp, and VH-Tlinker-VL is about 740bp.

[0033] 2. Antibody library screening

[0034] All clones after transformation were collected, induced by IPTG (0.25mmol / L) for 4h,...

Embodiment 2

[0037] Embodiment 2, preparation of scFv antibody

[0038] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0039] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0040] F1: 5'–CATG CCATGG GCCGTGACGTTGGACGAG-3';

[0041] R1: 5'–CCC AAGCTT TTAACCTAGGACGGTCAGGG-3'.

[0042] 3. Digest the PCR amplified product of step 2 with restriction endonucleases NcoI and HindIII, and recover the digested product.

[0043] 4. Digest the pET-27b(+) vector with restriction endonucleases NcoI and HindIII to recover a vector backbone of about 5367 bp.

[0044] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recombinant plasmid is described as follows: a double-stranded DNA molecule shown in ...

Embodiment 3

[0053] Embodiment 3, the preparation of VP2 protein

[0054] 1. Construction of recombinant plasmids

[0055] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing.

[0056] 2. Using the double-stranded DNA molecule synthesized in the step as a template, perform PCR amplification with a primer pair composed of F2 and R2 to obtain a PCR amplification product.

[0057] F2: 5'- GAAGAC TTAGGT ACAAACCTGCAAGATCAA-3';

[0058] R2: 5'- GGATCC TTA TGCTCCTGCAATCTTCAG-3'.

[0059] 3. The PCR amplified product of step 2 was double-digested with restriction endonucleases BbsI and BamHI, and the digested product was recovered.

[0060] 4. Digest the plasmid pHisSUMO with restriction endonucleases BbsI and BamHI to recover a vector backbone of about 5700 bp.

[0061] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. In the recombinant plasmid, the coding sequence of the VP2 protein coding...

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Abstract

The invention discloses a scFv antibody, an encoding gene thereof and application thereof to preparation of preparations for treating or preventing an infectious bursal disease. The scFv antibody is formed by a heavy chain variable region, a light chain variable region and a connecting region between the heavy chain variable region and the light chain variable region. The heavy chain variable region is (a) or (b), wherein (a) refers to protein which is formed by amino acid residues at the first site to the 124th site of the sequence 1, and (b) refers to protein which is subject to substitution and / or depletion and / or addition of the amino acid residues of (a) and has the same activity; and the light chain variable region is (c) or (d), wherein (c) refers to protein which is formed by amino acid residues at the 140th site to the 244th site of the sequence 1, and (d) refers to protein which is subject to substitution and / or depletion and / or addition of the amino acid residues of (c) and has the same activity. The scFv antibody provided by the invention has the advantages of high specificity, good curative effect and the like and creates a new situation in the prevention and treatment history of IBDV (Infectious Bursal Disease Virus), and even in the prevention and treatment history of the entire animal virus diseases.

Description

technical field [0001] The invention relates to a scFv antibody, its coding gene and its application in preparing preparations for treating or preventing chicken infectious bursal disease. Background technique [0002] Infectious Bursal Disease (IBD) is an acute and highly contagious chicken infectious disease caused by Infectious Bursal Disease Virus (IBDV). It is characterized by strong infectivity, high infection rate and high mortality rate. The disease has spread all over the world and is one of the most important diseases in the poultry industry, bringing huge economic losses to poultry enterprises. [0003] IBDV can multiply rapidly in the lymphocytes, especially B lymphocytes, in the chick's bursa of Fabricius, prompting the apoptosis of the B lymphocytes, causing the extreme atrophy of the bursa of Fabricius, and finally leading to immunosuppression. Antibody drugs are currently effective therapeutic drugs. Hyperimmune serum and egg yolk antibodies can have good e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21G01N33/569A61K39/42A61P31/14
Inventor 李德山李天鹤周兵李宁任桂萍尹杰超
Owner JIANGSU KANIONREAL BIOMEDICAL TECH CO LTD
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